Malignant hyperthermia (MH) is a hypermetabolic myopathy which is triggered in genetically-susceptible human and animal individuals by potent, volatile anesthetics such as halothane [Denborough and Lovell (1960) Lancet 2, 545: Harrison et al. (1968) Brit. Med. J. 3:594-595] or by depolarizing muscle relaxants such as succinycholine [Harrison et al. (1969) Brit. J. Anaesthesia 41:844-855]. In swine, it is also referred to as porcine stress syndrome [Topel (1968) Mod. Vet. Pract. 49:40-41; 59-60] because it may be triggered by exertional [Ludvigsen (1953) Internat. Vet. Congr. Stockholm 1:602-606], thermal [Forrest et al. (1968) J. of Appl. Physiol. 24:33-39], anoxic [Lister et al. (1970) Am. J. Physiol. 218:102-107], or mechanical [Gronert (1980) Anaesthesiol. 44:36-43] stressors as well as anesthesia [Hall et al (1966) Brit. Med. J. 4:1305]. A similar stress syndrome may occur in NH-susceptible (MHS) humans [Wingard (1974) Lancet 2;1450-1451] and dogs [O'Brien et al. (1983) Can. Vet. J. 24:172-177]. MH is characterized by the peracute development of contracture and maximal rate of metabolism in muscle. These have been proposed to occur due to an uncontrollable and sustained elevation of myoplasmic calcium [Britt and Kalow (1970) Can. Anesthetists Soc. J. 17:316-330: Lopez et al. (1985) Biophys. J. 47:313a], which is known to activate the contractile apparatus and metabolic machinery of skeletal muscle [Martonosi (1984) Physiol. Rev. 64:1240-1319].
The hyperactivity of muscle which occurs during MH results in the depletion of ATP and glycogen stores and the excessive formation of carbon dioxide, lactic acid and heat. This thermogenesis, in conjunction with peripheral vasoconstriction, leads to hyperthermia. The rapid rate of aerobic metabolism, by depleting blood oxygen, causes cyanosis [Gronert (1980) Anaesthesiol. 53: 395-423]. During MH, glycogenolysis, rhabdomyolysis and acidemia cause the release of large amounts of potassium from muscle and liver [Hall et al. (1980) Brit. J. Anaesthesiol. 52:11-17] into the vascular compartment. The resultant hyperkalemia contributes to the development of cardiac dysrhythmia and subsequent heart failure [Britt (1983) in "Complications in Anesthesiology" F. K. Orkin and L. H. Cooperman (eds) Lippincott, pp. 290-313].
Major revenue loss in the swine, pork and bacon industries occurs because of stress-induced MH deaths, usually during transport to the slaughterhouse. This occurs in homozygotes which make up 1 to 2% of swine and is referred to as porcine stress syndrome (PSS). Greater loss occurs due to the development of inferior quality meat after slaughter of MHS homozygote or heterozygote swine, which make up 10 to 30% of the population.
Activities and environmental stressors, which may trigger MH, include transport, restraint, mating, farrowing, fighting, vigorous exercising, and hot, humid weather [Mitchell and Heffron (1982) Adv. Food Res. 28:167-230]. The neural stimulation of muscle which occurs during slaughter [McLoughlin (1971) in "Condition and Meat Quality of Pigs" G. R. Hersel-de-heer et al (eds) Pudoc, pp 123-132], and the anoxia [Lister et al (1970) Am. J. Physiol. 215:102-107] which occurs with cardiac failure, are sufficient to trigger hypermetabolism in MHS muscle.
As a result of the excessive rates of production of lactic acid and heat [Lawrie (1960) J. Comp. Pathol. 70:273-295], sarcoplasmic proteins denature, thereby causing a deterioration of the water-binding capacity of muscle. Furthermore, the increased osmotic activity due to end-products of hypermetabolism causes an influx of water from the extracellular space, thereby resulting in hemoconcentration and increased intramyofiber water content [Berman et al (1970) Nature 220:653-655]. The muscle becomes pale, soft and exudative, sour-smelling and loose-textured [Briskey (1964) Adv. in Food Res. 13:89-178]. The shrinkage due to water loss during storage, transport and processing of the carcass is the major cause of wholesale losses at pork packing plants [Smith and Lesser (1982) Anim. Prod. 34:291-299]. Shorter shelf-life and decreased organoleptic acceptability contribute to retail losses. Other causes of lost revenue with MHS swine are their decreased average daily weight gain, conception rates, litter sizes and boar breeding performance [Webb (1980) Vet. Record 106:410-412; Carden et al (1985) Anim. Prod. 40:351-358].
In 1972, revenue loss in the United States due to porcine MH was estimated to be at least a quarter of a billion dollars annually [Hall (1972) Proc. of Pork Quality Symp. (R. G. Cassens et al eds) pp. ix-xii]. Since the late 1960s and the mid 1970s, the incidence of MH in North American swine populations is reported to have decreased approximately twofold [Grandin (1980) Internat. J. Studies in Anim. Product 1:313-317]. This decrease occurred as pig producers and meat packers modified their swine breeding programs and swine- and pork-management practices in order to decrease the incidence of MH. This was achieved by reducing the use of heavily muscled swine as breeding stock, reducing pre-slaughter stress on market hogs and increasing carcass chilling rates [Topel (1981) Proc. Work Planning Meeting of FSE/DSD, Ottawa, Agric. Can. 1-12].
Occurrence of MH is sporadic in all animal species except for swine, where, because of an association with heavy muscling, the disease has become endemic and in some countries has reached epidemic proportions [Webb (1980) Vet. Record 106:410-412; Mitchell and Heffron (1982) Ad. Food Res. 18:167-230]. In swine, the incidence of MH-susceptibility is breed and strain-dependent, ranging from less than 1% to greater than 90% of the herd. Increased prevalence of this disease, especially in European countries, is postulated to be a reflection of genetic improvement programs based entirely on performance and production parameters such as depth of back fat, muscularity, and carcass yields.
For Dutch and German strains of Pietrain and Poland China, and German and Belgium strains of Landrace swine, 68 to 94% of individuals were found to be MHS homozygotes and heterozygotes. By contrast, less than 10% of swine in North American herds are found to have susceptibility to MH. Because these figures are based on a diagnostic test (halothane challenge test) with poor sensitivity, they underestimate the incidence of pale, soft and exudative pork [Webb (1980; 1981) supra]. Furthermore, swine and carcass management practices at slaughter affect the incidence and degree of pale, soft, exudative pork from MHS swine [Topel (1981) supra]. On the other hand, these figures overestimate the occurrence (less than 1% in North America) of stress-induced MH-deaths, which may be largely eliminated by `stress-free` management practices [Topel (1981) supra].
In Canadian swine, the prevalence of MH gene homozygosity is up to 6% for Canadian Landrace and 1% for Yorkshire swine [D'allaire et al (1982) Can. Vet. J. 23: 168], the most important breeds. Based on recent data indicating that 1.9% of Ontario swine are homozygous for MH and a Mendelian inheritance pattern, the prevalence of heterozygotes would be 24%. Administration of succinycholine in the halothane challenge test detects many heterozygotes.
The high fatality rate of MH, the recognition of its heritability, and its association with inferior quality pork, have prompted the development of diagnostic tests for susceptibility to MH in swine. Halothane challenge testing is used extensively in the swine industry to detect susceptibility. Major limitations of this test, however, include its low sensitivity [Nelson et al. (1983) J. Anesthesia Analgesia 62:545-552; Seeler et al. (1983) supra; Webb et al. (1986) Anim. Prod. 42:275-279] and the high number of fatalities which may occur. In a typical test, two to three month old pigs are physically restrained and forced to inhale 3 to 5% halothane in oxygen through a face mask for several minutes. Those developing extensor muscle rigidity during the test are diagnosed as MHS. While the onset of MH signs is delayed by prior tranquillization [McGrath et al. (1981) Am. J. Vet. Res. 42:195-198], thermal, exertional, pharmacological, or psychological stresses will speed its onset [Van den Hende et al. (1976) Brit. J. Anaesthesia 48:821-829; Seeler et al. (1983) supra].
The caffeine [Kalow et al. (1970) supra] and halothane [Ellis et al (19-1) Brit. J. Anaesthesia 43: 721-722] contracture tests are considered to be the most definitive preanaesthetic diagnostic tests for MH-susceptibility [European Malignant Hyperpyrexia Group, 1984, Brit. J. Anaesthesia 57:983-9903. They are used extensively in man and have been applied successfully in swine [Gronert (1979) Anaesthesia Analgesia 58:367-371], horses [Waldron-Mease and Rosenberg (1979) Vet. Res. Commun. 3:45-50] and dogs [O'Brien et al. (1983) supra].
In veterinary medicine, contracture tests are seldom used because of cost, the trauma involved and the need for appropriate equipment and expertise. In these tests, muscle is excised, placed in oxygenated physiological saline, and connected to a force-displacement strain gauge. Isometric tension of the muscle is transduced into an electronic signal and recorded by a polygraph. To demonstrate its viability, the muscle is made to twitch continuously by applying electrical stimuli. Caffeine or halothane is then added to the bathing solution in increasing amounts. Muscle from MHS individuals is hypersensitive to the contracture-producing effects of these drugs. They increase the baseline tension of MHS muscle more, and at a lower concentration, than occurs for normal muscle [Britt, 1979, Int. Anaesthesia Clin. 17:63-96].
Diagnosis of MH-susceptibility may be facilitated by various indirect blood tests for MH, such as:
(1) haplotyping swine for the MH-gene-linked marker loci for H [Rasmusen and Christian (1976) Science 191:947-948) and S (Rasmusen (1981) Anim. Blood Groups in Biochem. Genet. 12:207-209] blood groups, erthyrocyte phosphohexose isomerase and 6-phosphogluconate dehydrogenase [Jorgensen et al. (1976) Acta Vet. Scand. 17:370-372], and serum protein postalbumin-2 [Juneja et al. (1983) Anim. Blood Groups in Biochem. Genet 14:27-36]; PA1 (2) erythrocyte osmotic fragility tests [King et al 1976, Ann. Genet. Select. Anim. 48:537-540]; PA1 (3) hyperlactatemia and hemoconcentration following intravenous injection of low dosages of halothane [Gregory and Wilkins (1984) I. Sci. Food Agric. 35:147-153]; PA1 (4) post-exertional alterations in blood parameters, such as hyperlactatemia, hemoconcentration, or elevated catecholamines [Ayling et al. (1985) Brit. J. Anaesthesia 57: 983-990; D'Allaire and DeRoth (1986) Can. J. Vet. Res. 50:78-83; Rand and O'Brien (1986) Am. J. Vet. Res. 190:1013-1014]; PA1 (5) enzymatic [Schanus et al. (1981) Prog. Clin. Biol. Res. 55, 323-336] and thermochemiluminescent [Kiel and Erwin (1984) Anal. Biochem. 143:231-236) assays for antioxidant system deficiency; PA1 (6) electron microscopic detection of hypertrophied open canalicular systems and energy dispersive microanalytic detection of decreased surface membrane calcium in platelets [Basrur et al. (1983) Scanning Elect. Microsc. 5:209-214]; PA1 (7) abnormal halothane-induced purine nucleotide profiles of platelet-rich plasma (Solomons and Masson (1984) Acta. Anaesth. Scand. 27: 349-355]; PA1 (8) abnormal halothane-induced increase in cytosolic calcium of isolated lymphocytes [Klip et al. (1986) Biochem. Cell Biol. 64:1181-1189]. PA1 (1) the Ca.sup.2+ release channel of the sarcoplasmic reticulum (ryanodine receptor) from MHS pigs has a higher affinity for ryanodine binding and requires higher concentration of calcium to inhibit ryanodine binding [Mickelson et al (1988) J. Biol. Chem. 263:9310-9315]; PA1 (2) patterns of tryptic digestion of the ryanodine receptor, as analyzed with a specific antibody, are altered in MHS swine [Knudson et al (1990) J. Biol. Chem. 265:2421-2424]; PA1 (3) in single channel recordings, MHN Ca.sup.2+ release channels were inactivated by pCa less than 4, whereas MHS channels remained open for significantly longer times, demonstrating an altered Ca.sup.2+ release channel inactivation [Fell et al (1990) Biophys J. 50:471-475] PA1 i) the cDNAs encoding a protein having 5035 amino acids and a molecular weight of approximately 564,000 daltons; PA1 ii) the DNA having a length of approximately 15.3 kb; and PA1 iii) isolated from chromosome 6 of pigs.
These tests lack sensitivity, specificity, reproducibility and/or have not been widely evaluated [Britt (1985) supra; Lee et al (1985) Anaesthesiol. 63:311-315; O'Brien et al. (1985) Am. J. Vet. Res. 46: 1451-1456; Webb (1981) supra].
Tests which assay for the hypermetabolic and/or degenerative changes occurring in `triggered` MH muscle include tests for ATP-depletion using in vitro biochemical assays [Harrison et al. (1969) Brit. J Anaesthesia 41:844-845] or in vivo 31-phosphorus nuclear magnetic resonance spectroscopy [Roberts et al. (1983) Anaesthesiol. 59:A230]; increased calcium efflux from isolated mitochondria [Cheah and Cheah (1981) Biochim. Biophys. Acta 634:40-49]; increased oxygen consumption and muscle twitch response following local ischemia [Jones et al. (1981) Anesthesia Analgesia 60:256-257] or decreased calcium uptake by histologic sections [Allen et al. (1980) Anesthesiol. 53:S251] or isolated sarcoplasmic reticulum [O'Brien (1986) Can. J. Vet Res. 50:329-337]. These tests have not been standardized nor widely evaluated. Furthermore, they may not be specific since they are indirect, measuring changes occurring secondarily to the underlying molecular defect.
A calcium-release sensitivity test performed on isolated muscle sarcoplasmic reticulum [O'Brien (1986) Can. J. Vet. Res. 50:318-329] is apparently a biochemical correlate of the physiological caffeine and halothane contracture tests, although it may be one to two orders of magnitude more sensitive.
Recently direct analysis of the Ca.sup.2+ release channel protein has revealed differences that may correlate directly with the disease:
Calcium-release sensitivity tests and tests of the structure and function of the ryanodine receptor have not yet been used for medical or commercial purposes.
The calcium release channel of the sarcoplasmic reticulum (ryanodine receptor) is a large protein that spans the gap between the transverse tubule and the sarcoplasmic reticulum. The channel is activated by ATP, calcium, caffeine, and micro-molar ryanodine and inhibited by ruthenium red, tetracaine, calmodulin, high Mg.sup.2+ and ryanodine (hence the name "ryanodine receptor") [Lai et al. (1988) Nature 331:315-319].
DNA encoding the human and rabbit skeletal muscle ryanodine receptors (RYR1) [Zorzato et al. (1990) J. Biol. Chem. 265:2244-2256] and the cardiac muscle ryanodine receptor (RYR2) [Otsu et al (1990) J. Biol. Chem. 265:13472-13483] has been cloned and sequenced. The deduced amino acid sequences of the RYR1 gene product comprise 5032 amino acids. The predicted protein structure suggests that the calcium release channel contains up to 12 transmembrane domains lying in the C-terminal portion of the molecule. The remainder of the protein is predicted to constitute the "foot" portion which spans the gap between the transverse tubule and the sarcoplasmic reticulum. Potential binding sites for calcium, calmodulin, ATP and other modulators of calcium channel function are also believed to be present in both RYR1 and RYR2 molecules between residues 2600 and 3000. Genetic linkage between the ryanodine receptor (RYR1) and MH genes has been determined in humans [MacLennan et al. (1990) Nature 343:359-361]. These observations strongly support the view that the ryanodine receptor is involved in malignant hyperthermia and that a defect in the calcium release channel might be the basic defect in animals, including humans, with MH.
The gene responsible for halothane sensitivity (HAL) has been found to segregate in pigs with a number of other genetic markers including S (S locus affecting expression of A-0 red blood antigens), Phi (glucose phosphate isomerase), H (H locus encoding blood group antigens), Po2 (postalbumin-2) and PgD (6-phosphogluconate dehydrogenase) [Rasmussen and Christian (1976) supra; Rasmussen (1981) supraJorgensen et al (1976) supra; Juneja et al (1983) supra]. It is, therefore, assumed that these genes are linked on pig chromosome 6pll-q21 [Horbitz et al. (1990) Genomics 8:243-248]. The human RYR1 gene has been localized to chromosome 19q13.1 and shown to be syntenic with these same genes [MacKenzie et al. (1990) Am. J. Hum. Genet. 46:1082-1089]. Linkage between the human RYR1 gene on chromosome lgq13.1 and the MH gene has been established in humans [MacLennan et al. (1990) supra].
We have discovered that the ryanodine receptor gene is the gene that is defective in porcine MH. This invention relates to the isolation and sequence analyses of cDNAs encoding normal and mutant porcine skeletal muscle sarcoplasmic reticulum calcium release channel proteins (alternatively referred to as ryanodine receptors) and determination that a mutation in the RYR1 gene in MHS strains segregates with the MH phenotype in pigs, such as Yorkshire, Pietrain, Landrace and Duroc strains and, predictably in all other heavily muscled strains (eg. Poland China; hampshire). The discovery that the porcine RYR1 gene is the gene responsible for MH and the discovery of diagnostic DNA probes from the porcine RYR1 gene forms the basis, according to an aspect of the invention, for a definitive DNA and/or antibody-based diagnostic test for MH.